38

The role of the cytoskeleton in polarity and rnorphogenesis of algal cells Diedrik Menzel
Selected algal species continue to serve as model organisms for the study of cell growth and cellular morphogenesis. Recent improvements in immunohistochemical and microinjection methods have helped to consolidate our views of the role of the cytoskeleton as a generator of spatial patterns in the cytoplasm before cellular morphogenesis. Progress has also been made in the discovery and characterization of molecular components of both the cytoskeleton and the extracellular matrix (ECM). Studies on the oocytes of fucoid brown algae have demonstrated that the ECM serves an active role in controlling cell shape and in defining the developmental fate of a cell. Actin, transmembrane proteins of the ~-integrin type, and vitronectin-like proteins in the ECM have been discussed as important elements in polar axis formation in the early steps of post-fertilization development. The mechanism of cell expansion has been investigated in the large coenocytic cells of the siphonoclad green algae. It was shown that the alignment of cell wall microfibrils in these cells depends on the degree of order in the cortical microtubule system. However, in contrast to earlier hypotheses, microtubules do not appear to function as physical boundaries guiding the paths of cellulose synthesizing terminal complexes in the plane of the plasma membrane. Recent work on the giant unicellular green alga Acetabularia has revealed dynamic reorganizations of the actin cytoskeleton during the course of apical morphogenesis. Actin has also been suggested to play a role, in more subtle ways, in the establishment of membrane prepatterns during cellular morphogenesis of the desmid green alga Micrasterias, prepatterns that predict regions of future surface expansion.

with great success as experimental model organisms for studying the deposition and modification of wall material, the structure and dynamics ofcytoskeletons, and the origin and maintenance of ion gradients. In this review, I will summarize recent results obtained from the study of these model organisms and discuss emerging concepts of cellular morphogenesis.
P o l a r i t y a n d initiation o f tip g r o w t h in Fucus zygotes

Once spatial order in the organization of cell components has been established, the mechanism of tip growth may be seen as a device to perpetuate the preset polarity. However, completely symmetrical cells such as the oocytes of the marine brown algae Fucus and Pelvetia need to determine a new axis of polarity, and rely on environmental cues for this purpose. Kropf [1] demonstrated the induction of polarity in fertilized eggs of Peivetia after unilateral illumination; induction of polarity resulted in rearrangements of the cortical actin cytoskeleton. T h e first visible sign of polarization was the formation of an actin cap at the pole of the egg opposite the light source. This region subsequently bulged out to form the rhizoidal pole. T h e radial array of microtubules (MTs) originating from the nuclear surface did not play a role in axis formation but contributed instead to a later stage of rhizoidal outgrowth and nuclear orientation [1]. T h e axis position is thought to be fixed in space by the anchorage of actin filaments to the membrane region of the presumptive rhizoidal pole via transmembrane protein complexes of the 13-integrin type. These protein complexes are presumed to mediate the connection between the actin cytoskeleton and an extracellular matrix (ECM) component. A homologue of animal 13 integrin has been discovered by immunological cross-reactivity, and its function in polar axis formation has been confirmed directly by microinjection of antil3-integrin antibodies, a process which results in the loss of polar axis formation [2]. A promising candidate for an ECM protein (which will interact with the I~-integrin-type protein complex) in Fucus is a 62kDa vitronectin homologue, which has been identified by immunological cross-reactivity with heterologous antivitronectin antibodies [3], and is immunolocalized in the tip of growing rhizoids [4]. Unexpectedly, ECM components in the fucoid species Pelvetia are capable of redirecting the protoplast's path of differentiation. This has been shown by elegant laser ablation experiments, which either allow one cell of the two-cell stage embryo to be destroyed [5], or can be used

.llilkl r a S S

Max-Planck Institute for Cell Biology, Rosenhof, D-68526 Ladenburg, Germany; e-mail: mp685dm@genius.embnet.dkfz-heidelberg.de

Current Opinion in Cell Biology 1996, 8:38-42

Current Biology Ltd ISSN 0955-0674

Abbreviations ECM extracellularmatrix MF microfilament MT microtubule

Introduction

Plant cells can grow and become shaped by uniform, as well as locally restricted, expansion of the cell wail, resulting in an unlimited variety of cell morphologies. For the study of cell morphogenesis, it has proven useful to turn to cell types that either display simple versions of localized cell expansion or become unusually large. Among the countless taxa of algae, some have been used

The role of the cytoskeletonin polarity and morphogenesisof algal cells Menzel

39

to free protoplasts from their respective wall casings [6]. If one cell is destroyed, the remaining cell does not change its differentiation course and the lost parts are not replaced [5]. On the other hand, a rhizoidal cell protoplast dedifferentiates and spontaneously repolarizes into both rhizoidal and thallus cells within 24 hours after treatment by laser ablation [6']. Hence morphogenetic omnipotence is apparently preserved in protoplasts. However, this omnipotence is overridden as soon as the protoplast comes into contact with cell wall remnants. Rhizoidal protoplasts in contact with the inner thallus wall differentiate in a thallus-like pattern, and protoplasts derived from thallus cells become rhizoid cells if they come into contact with the inner face of rhizoidal walls [6]. Homologues of 13 integrin and of spectrin, which is a component of the sub-membrane actin network in animal cells, have been implicated in the anchorage of actin filaments in the apical membrane patch of the phycomycete Saprolegnia [7]. These recent findings in algae and fungi reinforce the current opinion that similarities exist between animal ECMs and the walls of fungi and plants [8]. Hence, mechanisms that connect the cytoskeleton with the ECM, through the assistance of transmembrane proteins, are thought to be comparable in plants and animals [9]. E x p a n s i o n g r o w t h in g i a n t a l g a l cells Tip growth often occurs in conjunction with intercalary cell expansion or other modes of cell enlargement, resulting in either larger cells or variable cell morphologies [10]. Members of the green algal order Siphonocladales are excellent models for studies of cell expansion. T h e y present a cellulosic wall with an exquisite regularity in the order of microfilaments (MFs), in addition to a dense system of evenly spaced cortical MTs running from the tip for several tens of millimetres down to the base of the cell [11]. Because the MTs appear to be cross-linked to the plasma membrane and do not overlap one another, they form a spatial arrangement that comes closest to representing the fluid channel hypothesis; this hypothesis states that membrane-bound MTs provide the boundaries within which cellulose synthesizing terminal complexes (TCs) are restricted in their freedom of movement [12]. However, although there is general agreement that MTs are involved in controlling MF deposition in most higher plant cells [13], there is continuing controversy over whether this is true for cells growing exclusively by apical elongation (i.e. by tip growth) [14]. General features in the relationship between MTs and wall microfilaments have emerged from the work of Mizuta [11]. With slight differences between species, cell wall formation in the Siphonoclads proceeds in pulses from the tip to the base. During the process of lamella formation, TCs travel as a coherent wave front in the plane of the plasma membrane as they produce cellulose MFs. T h e interval between each new wave front being issued from

the tip is so short that, at any given time, several growing lamellae can be observed in the same cell. T h e orientation of fibrils within a given lamella is constant, but it alternates between helicoidal and crisscross patterns from one layer to the next [11,15]. T h e behaviour of MTs and MFs formed during the course of regeneration of protoplasts in siphonoclad algae strongly suggests a causal relationship between the two elements. In the absence of MTs, wall MF formation is absent, but recurs in a random fashion as MTs reappear. Concomitant with the partial alignment of newly formed M T arrays, a helicoidal pattern of MFs occurs in the new wall layer. Finally, after a new axial system of MTs has been re-established, crisscross patterns reappear and tip growth resumes [16]. Kimura and Mizuta [17] confirmed that a relationship exists between the density of MTs and the regularity in the MF pattern by studying cell wall formation in the presence of different concentrations of the M T inhibitor amiprophosmethyl (APM). A high density of MTs resulted in crisscross MF patterns, a medium density was responsible for helicoidal patterns and low densities corresponded to a random orientation of wall MFs. Resumption of growth, after recovery from treatment with APM, occurred along the original polarity axis provided that some of the original MTs were present in the cytoplasm [18]. However, when MTs were completely depolymerized, polarity information was lost. In such cells, random M T systems were formed initially; these systems eventually reorganized into converging radial arrays. Each radial array initiated the formation of a new growing apex, from which new axially oriented MTs were organized. From this evidence, it is clear that in the Siphonoclads ordered MF deposition and vectorial growth require polarized and highly ordered M T systems. However, the above observations do not support the fluid channel hypothesis. In addition, the fact that orientations of MFs alternate between layers, and that each layer is issued from the tip, points to the existence of a switching mechanism at the apex involving the converging M T array. Alternation of M T density could be a key element in the switching mechanism, and the recent discovery of microtubule-organizing centre (MTOC)-like activity in Chaetomorpha [19] indicates that, in this group of algae, molecular components exist that are capable of controlling such changes in M T dynamics. Siphonoclad cells contain a cortical actin filament system that can be induced to contract in a calcium/calmodulindependent fashion by wounding the cell [20]. The possibility of an involvement of this actin cytoskeleton in MF deposition has only recently been experimentally approached in these cells [21"]. Interestingly, it was found that the helicoidal MF orientation occurring in Boergesenia cells in the absence of MTs was sensitive to treatment with cytochalasin, an inhibitor of actin polymerization,

40

Cytoskeleton

suggesting that actin probably does play a role in MF patterns in an as yet undetermined way.

Apical morphogenesis in Acetabularia Acetabularia is a giant unicellular green alga with a polar, tube-like organization. The apex is capable of alternating between tip growth and whorl branch initiation [22]. In contrast to the situation in the Siphonocladales, cortical microtubules are absent. T h e cytoskeleton of Acetabularia consists of a system of parallel, axially orientated, cortical actin bundles, which support vigorous multistriate cytoplasmic streaming and run right to the tip where they converge into a dense, radial network. This actin configuration is associated with apical cell expansion [23].
As the cell prepares for whorl branch initiation, growth is transiently suspended, the tip flattens and the axial actin filament bundles at the tip disperse and become reorganized into a random network (H Sawitzky, J WillingaleTheune, D Menzel, unpublished data). Following the network stage, focal points of stronger actin staining appear around a subapical annulus. These points of actin staining mark small, localized lytic cavities in the cell wall that subsequently enlarge, giving rise to side branches. It is remarkable that, in contrast to regular tip growth, morphogenesis is initiated by localized lytic processes in the wall that occur apparently before new wall material is supplied and buds are formed. Calcium is important for tip growth and for the process of pattern formation that precedes whorl initiation. Higher concentrations of membrane-bound calcium appear first in the subapical annulus, and then concentrate in the lytic cavities [24]. The mechanisms undedying the redistribution of calcium in these cells is not known. Goodwin and Briere [25] have employed mathematical modelling to address the mechanical relationships between the shape of the apex and physical parameters defining the behaviour of cell wall and cytoplasm, such as strain, elasticity, plasticity and self-organizing ion gradients. By systematically changing these parameters in the simulation process, they arrived at patterns resembling those seen in nature, such as flattening of the tip and initiation of radial, symmetrically arranged growth buds. T h e reorganization of the actin cytoskeleton taking place during apical morphogenesis in Acetabularia is likely to depend on myosin. In theory, myosins could play a role in tipwards bound vesicle transport, maintenance of tension in the tip cytoskeleton, or signalling processes at the tip membrane [26]. Such functions have been discovered for MyoA, a class I myosin, in the ascomycete fungus Aspergillus, in which MyoA is found in the hyphal tip [27']. Myosin has also been found to be localized at the tips of pollen tubes by the use of specific antibodies against the recently characterized 170 kDa pollen myosin [28"], but the question of its function in tip growth has not been experimentally addressed.

Myosin has recently been discovered in Acetabularia, and two isoforms of the heavy chain of myosin have been characterized at the genetic level (O Vugrek, D Menzel, unpublished data). It was found that both of these myosin isoforms have very short carboxy-terminal tails and lack any potential coiled-coil regions, features which are typical of class I myosins. However, on the basis of phylogenetic sequence analysis, they are definitively not members of class I (D Menzel, unpublished data). Nevertheless, they could have a function equivalent to that of the Aspergillus MyoA.

Radial morphogenesis in Micrasterias

The freshwater desmid Micrastenas consists of two identical multilobed halves connected by an isthmus [29]. In mature cells, the nucleus is situated in the isthmus where it divides. After completion of mitosis the isthmus is 'closed', and each cell half creates an exact mirror image of itself. This process begins with the formation of a bud at the isthmus; the bud then gradually expands to the size of the mature cell half. During radial expansion of the young cell half, lobes form which split further into 'fingers'. Early pharmacological studies have suggested an involvement of actin filaments in growth and shaping of these lobes [29]. Recent attempts to directly visualize F-actin in the growing lobes of two species of Micrasterias, by microinjection of fluorescein isothiocyanate (FITC)-phalloidin, failed. However, in other regions of the cell, actin was strongly labelled [30]. An explanation could be that at the apex of the growing lobe actin is different from that in the remainder of the cell and, for unknown reasons, cannot be visualized by FITC-phalloidin binding. F-actin in pollen tubes can be stabilized by high pressure freeze fixation and subsequently examined by electron microscopy [31]. This method, however, has also failed to reveal actin-like, filamentous structures in the growing lobes of Micrasterias [32], even though the linear arrangement of secretory vesicles seen at the fusion sites in the growing lobes would seem to require some sort of guidance by a cytoskeletal element. T h e existence of inward-flowing calcium currents at the apices of the lobes was earlier implicated by the use of vibrating probe measurements [33]. However, although the microinjection of dextrane-coupled, ion-specific stains into tip-growing plant cells reveals a tip-high cytoplasmic calcium gradient [34-36], such a gradient is not found in the Micrasterias lobes [37"']. There is still good reason to believe that calcium could play a role in vesicle fusion, because earlier studies detected membrane-bound calcium by means of chlorotetracyclin (CTC)-fluorescence at the tips of the growing lobes but not in the mature lobes. As membrane patches characterized by CTC-fluorescence occur very early on in the development of the new cell half, and mark the regions of future growth, multilobe growth in Micrastenas may be governed by pattern formation in the membrane [29]. One could speculate that

The role of the cytoskeleton in polarity and morphogenesis of algal cells Menzel

41

these patterns lead to the formation of membrane patches that are specialized by their possession of different types of docking proteins accepting vesicle types carrying either material for wall expansion or material designed to stiffen the wall. T h e former type of membrane patch would become the growing lobes, the latter the clefts between the lobes. This hypothesis is supported by the occurrence of two types of vesicles, one rarer type that is directed to the cleft areas and another more abundant type that amasses in the growing tip apices [29]. This hypothesis could also explain the lack of structural evidence to support the idea that F-actin is present in the lobes, by assuming that the process of pattern formation could be so shortlived, and involve such an insignificant amount of actin filaments, that it has easily evaded all past attempts to visualize it.

analysis programmes, turns out to group with actins of the phycomycetes (D Menzel, unpublished data).

Acknowledgements
I would like to cordially thank Julia Willingale-Theune for many helpful suggestions in the course of the preparation of this article. Sincere thanks also go to Peter Traub for supporting our work on Aceta/n~laria. Financial support from the Deutsche Forschungsgemeinschaft is gratefully acknowledged.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest
oo of outstanding interest

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Kropf D: Cytoskeletel control of cell polarity in a plant zygote. Dev Biol 1994, 165:361-371. Shaw SL, Cluatreno R: Mlcrolnjectlon of antibodies to determine the role of cytoplasmlc protelns In the estebllshment of polarlty In Fucus. J Cell Biocham Suppl 21a, 1995:448. Ouatreno RS, Brian L, Aldridge J, Schultz T: Polar axls flxetlon In Fucus zygotes: components of the cytoskeleton and extrecellular m~rlx. Development Suppl 1, 1991:11 - I 6. Wagner VT, Brian L, Quatrano RS: Role of a vltronectln-llke molecule in embryo adhesion of the brown alga Fucus. Proc Natl Acad Sci USA 1992, 89:3644-3648. Kropf DL, Coffman HR, Kloareg B, Glenn P, Allen VW: Cell wall and rhlzold polarity in Pe/ve//a embryos. Day Biol 1993, 160:303-314. Berger F, Taylor A, Brownlee C: Cell fate determination by the cell wall In eady Fucus development. Science 1994,
263:1421-1423.

Conclusions
Features of tip growth and apical morphogenesis among algal cells share superficial similarities, suggesting that the underlying mechanisms have diverged in the course of algal evolution. However, one basic characteristic, common to most algal cells, is that actin plays a major role. Even in cases where prominent M T systems are clearly involved in the control of MF alignment, evidence indicates that actin may play a role at a very basic level of MF organization. T h e actin cytoskeleton appears to be involved in directing secretory vesicles to their target sites, as well as in controlling prepatterns in the plane of the plasma membrane. T h e nature of these patterns in terms of protein components is not yet clear. However, transmembrane proteins such as 13-integrin-like molecules and calcium channels appear to be required. As shown in Fucus oocytes, proteins of the ECM help to stabilize these patterns and exert control over the path of differentiation in a much more direct way than originally perceived. Future work will have to focus on the submembrane cytoskeleton and on the transmembrane mediators between the cytoskeleton and the ECM in order to understand this functional relationship. It is particularly important to characterize the ECM proteins at the molecular level. Even though this line of research is still in its infancy in algal cells, an expanding arsenal of molecular methods promises rapid progress in the near future. T h e availability of molecular information will also be useful for the identification of common or divergent features in the mechanisms of morphogenesis among the algal lineages. As already noted by Kropf [1], preliminary information pertaining to sequence characteristics of Fucus proteins seemed to support the interesting notion that the Fucus rhizoid cell has a lot more in common with the fungal tip cells than with other tip-growing systems. This notion has been strengthened recently by the determination of the sequence of a Fucus actin gene [38], which, in preliminary runs through phylogenetic sequence
3.

4.

5.

6. ee

Physical contact between protoplasts (produced by laser ablation) and cell wail remnants is sufficient to cause redifferentiation of protoplasta in the pattern of the wall donor cell. This is a very interesting observation, indicating that the ECM exerts an influence for directing cell development that is much more important than was previously envisaged. 7. Kaminskyj SGM, Heat IB: Integdn and speotdn homologues, and cytoplasmic adhesion in tip growth. J Ceil Sci 1995,
108:849-856.

Heterologous antibodies against animal ~ integrin recognize a Saprolegnia polypeptide of 120kDa under reducing conditions, and these antibodies label membrane patches along a shallow tip-high gradient by immunostalning. Retraction of the protoplast from the tip by plasmolysis causes the labelled material to stick to the wall. A 246 kDa homologue of spectrin also localizes at the tip, but, in contrast to the I~-integrin homologue, this protein retracts, together with the protoplast, when the cell is ptasmolyzed. 8. 9. Wyatt SE, Carpita NC: The plant cytoskeleton-cell-wall continuum. Trends Cell Bio11993, 3:413-417. Reuzeau C, Pont-Lezica RF: Comparing plant and animal extracellular motdx-cytoskleton connections - are they alike? Protoplasma 1995, 186:113-121. Menzel D: Cell architecture and cellular morphogenesls of eukaryotic algae (Chlorophyta). Prog Botany 1994, 55:1-38.
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coenocytlc green algae. Prog Phycol Res 1994, 10:55-95. 12. Giddings TH, Steehelin LA: Mlcrotubule-medieted control of mlcrofibdl deposition: a re-examination of the hypothesis. In The Cytoskeletal Basis ot Plant Growth and Form. Edited by Lloyd CW. San Diego: Academic Press; 1991:85-100. Cyr JR: Mlcrotubules in plant morphogenesls: role of the cortical array. Annu Ray Cell Bio11994, 10:153-180. Emons AMC, Derksen J, Sassen MMA: Do mlcrotubules orient plant cell wall microfibdls? Physiol Plant 1992, 84:486-493. Okuda K, Matsuo K, Mizuta S: Charactedstics of the deposition of microfibdls dudng the formation of the polylamellate walls In the coenocytic green alga Chemeedoris orientalis, Plant Cell Physiol 1990, 31:357-364. Mizuta S, Katoh S, Harada T, Yamada H, Okuda K, Morinaga T: Involvement of mlcrotubules in microfibrlllar patterns In the

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cell walls of the developing coenecytic green alga Bood/es coecte. Botan/ca Marina 1991, 34:41 ?-424. 17. Kimura S, Mizuta S: Role of the mlcrotubule cytoskeleton In alternating changes In cellulose-microfibrll orlentetion in the coenocytlc green alga, Cheetomorpoha moniligere- Plants 1994, 193:21-31. Okuda K, Matsuo K, Mizuta S: The meridional arrangement of cortical mlcrotubules defines the site of tip growth in the ccenecytic green alga, Chemeedoris orient*lie. Botanica Marina 1993, 36:53-62.

18.

McGoldrick CA, Gruver C, May GS: MyoA of Aspergillus nidulans encodes an essential myosln-I required for secretion and polarized growth. J Cell Biol 1995, 128:577-587. myoA encodes a myo6in heavy chain with significant sequence homology to class I myosina. The authors of this paper have made a specific antibody against the MyoA protein, and found staining at the hyphal tip by using immunofluorascence micro~-..opy. They further provide compelling evidence for a function of MyoA in tip growth by disrupting myoA. 28. Yokota E, McDonald AR, Liu B, Shimmen T, Palevitz BA: LocallzeUon of a 170 kDa myosin heavy-chain in plant cells. Protoplasms 1995, 185:178-187. In a previous study, the authors of this paper used biochemical and in vitro motility assays to characterize a 170 kDa myosin heavy chain from plant cells. Here, they show that an antibody made against this new plant myosin labels organelle8 and the tip cytoplasm in pollen tubes. 29. 30. Meindl U: Micrasterles as a model system for research on morphogenesls. Microbiol Rev 1993, 57:415-433. Meindl U, Zhang D, Hepler PK: Actin mlcroftlaments are associated with the migrating nucleus and the cell cortex In the green alga Mictasterles-studles on living cells. J Cell Sci 1994, 107:1929-1934. Lancelle SA, Hepler PK: Ultrastructure of freeze-substituted pollen tubes of Lilllum Iongiflorum. Protoplasms 1992, 167:215-230. Meindl U, Lancelle S, Hepler PK: Vesicle production and fusion during lobe formation in Mlcrasterles visualized by highpressure freeze fixation. Protoplasma 1992, 170:104-114. Troxell CL, Scheffey C: Ionic currents flow through MIcrasterles and Closterlum cells during cell expansion of the pdmary cell wall. Plants 1991, 184:218-225. Berger F, Brownlee C: Ratio confocal Imaging of free cytoplasmic calcium gradients in poladsing and polarlsed Fucus zygotes. Zygote 1993, 1:9-15. Jackson SL, Heath IB: Roles of calcium ions In hyphal tip growth. Microbiol Rev 1993, 57:367-382.

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Mizuta S, Kaneko M, Tsurumi S: Assembly of cortical microtubules during cold treatment of the ccenocytlc green alga, Cheetomorpha moniligere. P/ants 1995, 196:190-192. Cold treatment causes loss of 70-75% of the cortical MTs; continuing cold treatment causes initiation of new short MTs from the sides of the cold-stable MTs, suggesting the presence of microtubule-organizing centrea and their relocation by cold treatment. 20. La Claire JW: Contractile movements In the algae: the slphonecisdalse as model systems. In The Cytoskeleton of the Algae. Edited by Menzel D. Boca Raton: CRC Press; 1992:240-253.

31.

Mizuta S, Watanabe A, Kimura S, Yoshida K: Possible Involvement of membrane fluidity In helicoidal mlcrofibdllar orientation in the coenocytic green alga, Boergesenia forbes#. Protoplasms 1994, 180:82-91. For many years, emphasis has been placed on the MTs as possible control elements in the orientation of wall MFs. The authors of this paper provide the first evidence that helicoidal MF patterns could be controlled by membranebound F-actin. 22. 23. Manzel D: Cell differentiation and the cytoskelaton in Acetabularle- New Phyto/1994, 128:369-393. Manzel D, Jonitz H, Elsner-Menzel C: The cytoskelaton in the life cycle of Acetabulerla and other related species of dasydad green algae. In The Cytoskeleton of the Algae. Edited by Menzel D. Boca Raton: CRC Press; 1992:195-217. Harrison LG, Graham KT, Lakowski BC: Calcium localization dudng AcetebuleHe whorl f o r m a t i o n - evidence supporting 8 2-stege hierarchical mechanism. Development 1988, 104:255-262. Goodwin BC, Brier, C: A mathematical model of cytoskeletel dynamics and morphogenesls in Acetsbulede- In The Cytoskeleton of the Algae. Edited by Menzel D. Boca Raton: CRC Press; 1992:219-238. Haason T, Mooasker MS: Molecular motors, membrane movements and physiology: emerging roles for myoslns. Curr Opin Cell Bio11995, 7:587-594.

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Miller DD, Callahan DA, Gross DJ, Hepler PK: Free Ca2+ gradient In growing pollen tubules of Ulium. J Ceil Sci 1992, 101:7-12. 37. Holzinger A, Callaham DA, Hepler PK, Meindl U: Free calcium ** in Micresteries-~ocal gradients are not detected In growing lobes. Eur J Cell Bio11995, 87:363-371. Microinjection of calcium green-dextren does not suggest the existence of a gradient of cytoplasmic calcium along the axis of a growing lobe, despite earlier observations that calcium currents enter the tip of the lobe and that membrane-bound calcium localizes at the tip. 38. Goodner BW, Davis JD, Quatrano RS: Sequence of 8ctln cdn8 from Fucus disticus. Plant Physio/1995, 107:1007-1008.